Vol. 55, Issue 3, 508-514, March 1999

Potency and Mechanism of Action of E4021, a Type 5 Phosphodiesterase Isozyme-Selective Inhibitor, on the Photoreceptor Phosphodiesterase Depend on the State of Activation of the Enzyme

Marc R. D'Amours, Alexey E. Granovsky, Nikolai O. Artemyev, and Rick H. Cote

Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire (M.R.D., R.H.C.), and Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa (A.E.G., N.O.A.)

The ability of inhibitors selective for the type 5 phosphodiesterase isozyme (PDE5) to act on the photoreceptor PDE isozyme (PDE6, the central effector enzyme for visual transduction) is poorly understood. Because PDE5 inhibitors are currently used as therapeutic agents, it is important to assess the potency and mechanism of action of this class of PDE inhibitor on PDE6. We show that E4021 (sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-yl]piperidine-4-carboxylate sesquihydrate) inhibits activated PDE6 (K_I = 1.7 nM) as potently as PDE5. This makes E4021 the most potent inhibitor of PDE6 discovered to date. The effectiveness of E4021 to inhibit nonactivated PDE6 (with bound inhibitory  subunits) is reduced 40-fold compared with the activated enzyme. Furthermore, at intermediate E4021 concentrations and high cGMP concentrations, nonactivated PDE undergoes activation of cGMP hydrolysis rather than inhibition. We demonstrate direct competition of E4021 and the  subunits for binding to the catalytic site. Measurements of cGMP binding to noncatalytic regulatory sites on the catalytic subunits of PDE6 rule out an allosteric effect of E4021 by direct binding to these noncatalytic sites. We conclude that E4021 is a competitive inhibitor of cGMP hydrolysis and that the  subunit also competes with both E4021 and substrate for catalytic site binding. An understanding of the effects of PDE5-targeted drugs on retinal PDE6 requires a knowledge of the complex interactions among substrate, drug, and inhibitory  subunit at the catalytic site of both nonactivated and activated forms of PDE6.